GnRH is a hypothalmic hormone which triggers the release of the gonadotropic hormones, particularly leutinizing hormone. These hormones regulate the functioning of the gonads to produce testosterone in the testes and estrogen in the ovaries; and they also regulate the production and maturation of gametes. Mammalian GnRH is a well known decapeptide as described in U.S. Pat. No. 4,072,668.
GnRH peptide analogs which are antagonistic to endogeneous GnRH and inhibit the secretion of gonadotropins and the release of steroids by the gonads are known from U.S. Pat. Nos. 5,506,207 and 5,169,932. These compounds are analogs of the decapeptide GnRH which include modified amino acids in the 5 and 6 position. Particularly useful are the peptide analogs which have the general formula: EQU Ac-.beta.-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA.sub.5 -D-AA.sub.6 -Leu-Lys(isopropyl)-Pro-D-Ala-NH.sub.2 (SEQ ID No:1)
wherein AA.sub.5 and AA.sub.6 are residues of amino-substituted phenylalanine (or its equivalent) where the substituent is preferably in the 4-position and is preferably an acylating agent of 5 carbon atoms or less or one which contains an unsaturated heterocyclic ring containing from 2 to 4 nitrogen atoms. Particularly useful is the GnRH peptide analog known as Azaline B, which contains an "Aph(Atz)" substituent, that is, a 5 membered triazole ring (specifically, a 1,2,4-triazole) as the substituent in the 4 position of the amino substituted phenylalanine of both AA.sub.5 and AA.sub.6. Also useful are the pepuides known as Azaline C, which has a methyl group on the backbone nitrogen of residue 5, and Acyline, which has an acetamido group as the substituent in the 4 position of the substituted phenylalanine. Further useful GnRH peptide analogues are reported by Rivier et al, J.Med.Chem, 1995, 38, 2649-2662, wherein the amino substituent is acylated with a simple amino acid residue, e.g. Gly, Ser, Ala, etc, followed by deprotection and triazole construction.
Previously, as described in the patents mentioned above, these GnRH analogs have generally been prepared by a classical solid phase peptide synthesis (SPPS) in which the peptide chain is elongated via sequential addition of amino acids to the resin-bound C-terminal amino acid by reaction with an excess of an appropriately activated and protected amino acid. In this method, to maximize coupling efficiencies and reduce side reactions, all couplings are performed with a minimal 3-5 fold excess of activated amino acid, coupling reagents and additives and all reactive side chain functionalities must be protected throughout the entire synthesis. In Azaline B, the key amino-triazoles on the phenylalanine residues of AA.sub.5 and AA.sub.6 are generally constructed only after the entire backbone has been assembled and immediately prior to global deprotection with concomitant resin cleavage. In one exception to this Rivier et al, J.Med.Chem, 1995, 38, 2649-2662, reports the construction of the amino-triazole units simultaneously on the protected resin anchored [5-10] hexapeptide followed by incorporation of the remaining 4 amino acids in the usual solid-phase synthesis manner.
While this SPPS procedure provides the GnRH peptides in acceptable yield and purity, the SPPS procedure exhibits several inherent limitations which make it undesirable for large scale production of the peptides. These include:
capacity of the resin which limits the amount of the final product; PA1 expense attributable to the C-terminal amide cleavable resins and the large excesses of coupling reagents, additives, and amino acids required; PA1 necessity of full protection for all reactive side chains as in the hydroxy group in Ser.sup.4, the aromatic amino groups in Aph.sup.5, D-Aph.sup.6 and the .epsilon.-i-propylamino group in Lys(i-Pr).sup.8 ; PA1 strongly acidic conditions required to affect global deprotection and cleavage of the peptide from the resin. PA1 (a) reacting a peptide of the formula: EQU T-(R.sub.2)AA.sub.5 -AA.sub.6 -X PA1 where T is (P.sub.2)AA4 or P.sub.2 and X is AA.sub.7 -OH or is --OH, with a peptide of the formula: EQU X'-AA.sub.8 -Pro-AA.sub.10 -NH.sub.2 PA1 or acid-addition salt form thereof, where X' is AA.sub.7 when X is absent and X' is absent when X is AA.sub.7 -OH; PA1 in a liquid reaction medium in the presence of a peptide coupling reagent and a stron organic amine base to obtain a product of the formula: EQU T-(R.sub.2)AA.sub.5 -AA.sub.6 -AA.sub.7 -AA.sub.8 -Pro-AA.sub.10 -NH.sub.2 PA1 (b) removing the P.sub.2 protecting group at the N-terminus, and PA1 (c) reacting the product of step (b) or an acid addition salt thereof, with a peptide of the formula: EQU G-AA.sub.1 -(R.sub.1)D-Phe-AA.sub.3 -T' PA1 or acid-addition salt form thereof, where T' is AA.sub.4 -OH when T is absent and is absent when T is P.sub.2 -AA.sub.4, in a liquid reaction medium to obtain a GnRH peptide of the formula: EQU G-AA.sub.1 -(A)D-Phe-AA.sub.3 -AA.sub.4 -(R.sub.2)AA.sub.5 -AA.sub.6 -AA.sub.7 -AA.sub.8 -Pro-AA.sub.10 -NH.sub.2. (SEQ. ID NO. 3) PA1 production of final product is limited only by vessel capacity thus permitting large scale bulk production; PA1 considerable savings can be realized by using stoichiometic or minimal excesses of required amino acids, coupling reagents, additives and reactants; PA1 provides carefully controlled coupling chemistry which reduces unwanted side reactions or eliminates the necessity for any side chain protection; PA1 the final product is isolated directly by precipitation eliminating the necessity for lyophilization. PA1 a) reacting a compound of the formula P.sub.2 -p-NO.sub.2 -PheOH with p-NO.sub.2 -PheOR where R is alkoxy, or an acid addition salt thereof, in the presence of a suitable peptide coupling reagent in a suitable solvent to obtain a dipeptide of the formula: ##STR2## PA1 b) reducing the dipeptide to obtain a compound of the formula: ##STR3## PA1 c) reacting the product of step b) first with diphenyl cyanocarbonimidate or an equivalent reagent and then with hydrazine in a suitable solvent to yield a compound of the formula: ##STR4##
In addition, because each amino acid is incorporated in a separate reaction, any side reaction or decoupling of the peptide in the later stages requires the process to be repeated from the beginning. Accordingly, there is a need for an improved process for the preparation of GnRH peptide analogs which is amenable to large scale manufacture. Other methods for the bulk scale production of peptides include liquid phase peptide synthesis (LPPS), enzymatic synthesis, or fermentation with genetically manipulated microorganisms. However, none of these methods have been applied to the production of GnRH peptide analogs.